Standard techniques of biotechnology permit the expression of desired sequences in suitable host cells by providing control sequences operable in these hosts. Specifically, if the production of a particular protein is desired in a bacterial host, the coding sequences for that protein are linked to bacterial promoter and ribosome binding site encoding sequences. Similarly, expression in eucaryotic hosts can be obtained by linking the coding sequence to, for example, yeast derived control sequences for expression in yeast, or to promoters derived from viruses normally infecting mammalian hosts to effect mammalian host cell expression.
While it has been possible to culture bacterial hosts transformed with vectors which effect expression to produce high levels of foreign proteins--sometimes corresponding to as much as 50% or more of total cell protein, eucaryotic recombinant hosts, in general, are not so productive. The presently available control systems are derived from viral sources, and ligated upstream from desired coding sequences for use in these cells. The eucaryotic differentiated transformants provide production levels which are several orders of magnitude lower than those obtained for the specialized products of these cells in vivo. This result is true despite the fact that differentiated cells may produce extremely large amounts of specialized products, endogenously. Erythrocytes for example, produce and secrete hemoglobin in amounts approaching 90% of total cell protein; pancreatic cells secrete amylase in total amounts approximating their cell mass. Protein production by recombinant eucaryotes is substantially lower in amount.
It is often desirable, nevertheless, to use eucaryotic, including mammalian, hosts for protein production since the processing effected by these cells, including e.g., glycosylation and/or proper folding of the protein products, may be a needed accompaniment to production of the peptide sequence. TPA and Factor VIII are examples of such glycosylated proteins. Thus, a method to increase the level of production of a desired gene product in cell cultures of eucaryotic cells would be of immense practical benefit.
Techniques using recombinant DNA also offer the possibility of host cell modification, i.e., it is often feasible to confer on the host cell some desirable property either with respect to the survival of the organism or, in the case of isolated cell cultures, with respect to maximizing an enzyme activity needed for the production of a secondary product. For example, increases in the level of enzymes responsible for the production of alcohol in yeast would improve the properties of the host organism as an industrial tool. In an example perhaps more relevant to the cell-specific sequences of the invention, bone marrow cells used for transplant might be encouraged to produce large amounts of immunosuppressant directed against T cells. In this context, also, regulation or enhancement of particular coding sequence expression would be advantageous.
Two major approaches have been taken to increase the level of production of a desired protein in eucaryotic hosts. In one, the desired coding sequence is ligated to a sequence which is capable of being amplified under certain selective conditions. The best known example of such an approach employs the sequences encoding dihydrofolate reductase (DHFR) which are amplified in the presence of the inhibiting drug methotrexate. In the second approach, expression constructs are provided with a viral-derived control element located 100-300 bp upstream from the transcription start site in the virus, which has been designated an "enhancer". The enhancers are short, cis-acting sequences which cannot themselves initiate transcription, but can potentiate transcription from a variety of promoters. Their effectiveness is relatively independent of position in the vector and of orientation. Such enhancers apparently operate in a manner which is not predictably dependent on the nature of the host cell. For example, the polyoma enhancer is functional in any differentiated mouse cell but not in undifferentiated embryonic cells.
It is recognized that sequences associated with a native gene may show some preference for transformant hosts which represent cells to which the gene is endogenous. For example, myeloma cell transformants, produce higher levels of immunoglobulin K chain than do correspondingly transformed fibroblasts (Oi, V., et al, Proc Natl Acad Sci (USA) (1983) 80:825), and lens cells microinjected with the chick delta crystalline gene produce more of this protein than do similarly injected fibroblasts (Kondoh, H., et al, Nature (1983) 301:440).
Some progress has been made in identifying the DNA sequences associated with this specificity. It has recently been shown that an intron sequence in the coding region for immunoglobulin heavy chain contains a lymphocyte specific enhancer which apparently results in higher levels of heavy chain production in B-lymphocyte derived (myeloma) cells than in other transformants such as HeLa cells. The enhancing effect of this intron sequence was also studied with respect to production of SV40 T-antigen under control of the SV40 promoter (Banerji, et al, Cell (1983) 33:279) and shown to be effective in regulating expression of the T-antigen gene in lymphocyte derived myeloma cells, but not in HeLa cells. It has also been shown that DNA sequences from the J-C region of the immunoglobulin gene are cis-acting independently of orientation and are active in mouse B cells but not in mouse fibroblasts (Gillies, S. D., et al, Cell (1983) 33:717). See also Queen, C, et al, Cell (1983) 33:741.
If cell-specific DNA sequences could be employed so as to increase expression of foreign coding sequences to a level comparable to that attained for sequences natively associated with specialized cells, a quantum improvement in production of foreign proteins could be achieved. However, none of the foregoing DNA sequences provide a portable enhancer fragment which can be relied upon to increase the level of protein production in a particular cell type transformant host to such levels. Thus, while the possibility exists that genes native to differentiated eucaryotic cells may contain control sequences which are cell-specific, and which are supplementary to the usually considered requirements for expression such as promoter, polyadenylation signal, etc., no reliable source for such cell-specific enhancing sequences has been found. The present invention provides such sequences which can be used to effect enhanced expression in specific desired transformant host cell types.